The current method for sequencing DNA involves a number of costly reagents such as fluorescent ddXTPs, dXTPs, primers and polymerase. This method requires sophisticated equipment, which needs to be operated by a qualified technician. Also, this method is limited to sequences of less than one thousand nucleotides in length.
Other sequencing methods have been considered in order to reduce cost, simplify the method, and allow sequencing to take place out of the lab. Cycle extension, polymerase reading, exonuclease sequencing, and DNA micro-arrays are methods that have been considered (Braslaysky, I., B. Herbert, et al. (2003), PNAS 100(7): 3960-3964). These methods have been comprehensively reviewed (Marziali, A. and M. Akeson (2001), Ann. Rev. Biomed. Eng. 3: 195-223).
One potential method of sequencing DNA is based on threading a single strand of DNA through a nanopore and identifying its sequence from the variation in the ionic current flowing through the pore as the strand is threaded (Kasianowicz, J. J., E. Brandin, et al. (1996), Proc. Natl. Acad. Sci. 93: 13770-13773). A second potential approach is exonuclease sequencing (Chan, E. Y. (2005), Mutat. Res. 573: 13-40). This method involves digesting the DNA one nucleotide at a time (Dapprich, J. (1999), Cytomet. 36: 163-168; and Matsuura, S.-I., J. Komatsu, et al. (2001), Nuc. Ac. Res. 29(16): e79) and then identifying each of the released nucleotides. However, these methods require modification of the DNA before digestion or modification of the nucleotides once they have been released from the DNA by exonuclease. The development of exonuclease sequencing is currently being held back by the difficulty in identifying the nucleotides at the single molecular level as they are released by the enzyme. Investigators have tried to identify the nucleotides using fluorescent labeling with limited success.
Stochastic sensing involves placing a nanometer sized pore in an insulating lipid bilayer membrane and measuring the ionic transport through the pore. When an analyte interacts with a binding site within the pore, a change in the ionic current is detected (Braha, O., B. Walker, et al. (1997), Chem. & Biol. 4: 497-505; and Bayley, H. and P. S. Cremer (2001), Nature 413: 226-230). The extent and duration of the current block resulting from each binding event can reveal the identity of the analyte. The frequency of the binding events can reveal the analyte concentration. Various binding sites can be created within the pore by way of protein mutation, chemical modification, and by use of molecular adaptors and carriers (Gu, L.-Q., O. Braha, et al. (1999), Nature 398: 686-690; and Braha, O., J. Webb, et al. (2005), Chem. Phys. Chem. 6: 889-892).